Staphylococcus aureus (S. aureus) is a facultative anaerobic, gram positive, spherical bacterium considered to be an opportunistic pathogen. S. aureus commonly colonizes the nose, skin and mucosal surfaces of the gastrointestinal tract of healthy humans. Approximately 20-30% of the population is colonized with S. aureus at any given time. These bacteria often cause minor infections, such as pimples and boils in healthy individuals. Normally, mucosal and epidermal barriers (skin) protect against S. aureus infections. Interruption of these natural barriers as a result of injuries—such as burns, trauma or surgical procedures—dramatically increases the risk of infection and could cause severe and/or systemic infections. Diseases that compromise the immune system (e.g., diabetes, end-stage renal disease, cancer, AIDS and other viral infections), but also immunosuppressive therapies—e.g. as radiation, chemotherapeutic and transplantation therapies-increase the risk of infection. Opportunistic S. aureus infections can become quite serious, causing endocarditis, bacteremia, osteomyelitis and abscess formation, which might result in severe morbidity or mortality. S. aureus infections may be divided in localized infection, such as pneumonia, and clinically more complex S. aureus infections, such as blood stream infections and abscess formation caused by distant organ seeding.
S. aureus is a leading cause of bloodstream, skin, soft tissue, and lower respiratory tract infections worldwide. The frequencies of both nosocomial and community-acquired infections have increased steadily over the years. In addition, treatment of these infections has become more challenging due to the emergence of multi-drug resistant strains. In developed countries such as the United States, resistance to β-lactam antibiotics in methicillin-resistant S. aureus strains (MRSA) is a major problem in hospitals and other healthcare settings. Notably, the incidence rate of all invasive MRSA infections, including those outside of hospitals, is high compared with other bacterial pathogens and 20% of these infections result in death. In addition the occurrence of acquired resistance to vancomycin further limited the treatment options for severe S. aureus infections.
S. aureus has a diverse arsenal of virulence factors that contribute to the pathogenesis of disease. These can be broadly subdivided into surface and extracellular secreted proteins. Surface proteins include both structural components of the bacterial cell wall, such as peptidoglycan and lipoteichoic acid, and surface proteins preferentially expressed during exponential growth, including protein A, fibronectin-binding protein and clumping factor. Secreted proteins are generally expelled from the bacterial cells during the stationary phase of bacterial growth and include several toxins such as alpha-toxin (also known as hemolysin alpha), enterotoxin B, leukocidins (including Panton-Valentine Leukocidine PVL), lipase and V8 protease. Yet despite the broad knowledge about the biochemical and molecular properties of these toxins, the precise role of the toxins in the pathogenesis of S. aureus infections is not entirely understood.
Experimental evidence and epidemiological data have suggested that amongst other cytotoxins, alpha-toxin may be involved in the pathogenesis of pneumonia (Mc Elroy et al, 1999). Alpha-toxin is thought to engage surface receptors of sensitive host cells and thus attaching to the cell surface. This event promotes toxin oligomerization into a heptameric pre-pore and insertion of a β-barrel structure with a 2-nm pore diameter into the plasma membrane. The formation of the pore is causing loss of membrane integrity, destabilizing the cells and ultimately leading to apoptosis and cell lysis. In particular lymphocytes, macrophages, alveolar epithelial cells, pulmonary endothelium, and erythrocytes are sensitive to pore formation by alpha-toxin; however, granulocytes and fibroblasts appear resistant to lysis (McElroy et al., 1999).
The exact role of alpha-toxin in the inflammatory response and the induction of innate immune response to bacterial infections are not fully understood. S. aureus expresses a number of other virulence factors and to date the contribution of each virulence factor to disease manifestation is not fully understood and poses a challenge to the development of prophylaxis and therapy of clinically complex S. aureus infection.
Alpha-toxin is known to be one of the virulence factors for the establishment of S. aureus infections in the host and a number of studies have highlighted the importance of alpha-toxin in disease. e.g. instillation of purified alpha-toxin into rabbit or rat lung tissue triggers vascular leakage and pulmonary hypertension, which has been attributed to the release of different signaling molecules (e.g., phosphatidyl inositol, nitric oxide, prostanoids, and thromboxane A2). In the literature it has been shown that anti-alpha-toxin immunity is protective against the toxin's detrimental effects, but designing vaccines against alpha-toxin remains a significant challenge.
Wardenburg and Schneewind (2008) demonstrated that the severity of lung disease in mice correlates with the levels of alpha-toxin produced by a particular S. aureus isolate. Furthermore the authors showed that immunization against a nonpore-forming alpha-toxin variant induced immunity to pneumonia caused by S. aureus. These findings are consistent with a study from the same group demonstrating that alpha-toxin is important for the pathogenesis of CA-MRSA pneumonia (community-associated methicillin-resistant S. aureus). In another setting the authors demonstrated that antibodies against alpha-toxin also protected human lung epithelial cells from S. aureus-induced lysis (Wardenburg and Schneewind (2008)).
Although these results indicate that alpha-toxin contributes to lung tissue destruction, it is not yet clear whether the animals' death in the above described experiments resulted from direct destruction of lung cells by the toxin, from an excessive inflammatory response, or from both. Passive transfer of alpha-toxin antibodies significantly reduced circulating levels of interleukin 1β, a cytokine known to accompany acute lung injury. Therefore, it is reasonable to conclude that the inflammatory response may contribute to alpha-toxin-mediated lung damage.
During a localized infection such as pneumonia in humans, approx. 40% of patients with S. aureus pneumonia develop blood stream infections and disseminated disease. The dissemination of the bacterial infection can lead to blood stream infection and distant organ seeding. The blood stream infection can lead to septicemia, a rapidly progressing and frequently fatal complication of S. aureus infections.
The dissemination of an S. aureus infection is also commonly seen in S. aureus pneumonia animal models, again with approximately 40% of animal developing disseminated bacteremia due to tissue damage and spreading of the infection through epithelial layers into the blood stream and lymphatic tissue. Nevertheless the dissemination largely depends on the genetic background of the animal strain used and the potential of the innate immune system, such as neutrophil activation, to control the growth. e.g. neutrophil depleted C57B/L animals are highly susceptible to kidney infections with S. aureus whereas immune competent animals are resistant to infections. In contrast A/J animals were very susceptible, mainly due to delayed recruitment of neutrophils to the kidney (von Köckritz-Blickwede, 2008).
Although data on structure and function of S. aureus proteins became more comprehensive the development of an effective vaccine remains a challenge.
An attempt was made to safely confer immunity to alpha-toxin and S. aureus bacteria by the use of compositions comprising a combination of antibodies that specifically bind to an S. aureus alpha-toxin antigen and antibodies that specifically bind to another bacterial antigen (WO 2007/145689). These compositions, while comprising amounts of antibody that are not effective on their own, nevertheless neutralize infection and/or provide protection against infection by the synergistic activity of the combination of antibodies.
The protective efficacy of said combination of S. aureus toxin-neutralizing and opsonic antibodies at 72 hours post-bacterial challenge with a S. aureus isolate is demonstrated as compared to the protective effect of immunization with either neutralizing or opsonic antibodies alone. The combination of the opsonic and toxin-neutralizing antibodies demonstrated a protective effect in preventing skin and soft tissue infection and organ seeding. However, the neutralizing anti-alpha-toxin antibody disclosed by that patent application itself is not sufficient to prevent organ seeding/abscess formation or to neutralize infection.
A further attempt was made by Heveker et al (1994a, 1994b) that describes neutralizing human and murine monoclonal antibodies directed against S. aureus alpha-toxin. The human monoclonal antibody of IgG/lambda subtype is characterized by sequence and shows neutralization.
The anti-alpha toxin antibody producing human hybridoma described by Heveker (1994a) was isolated using peripheral blood leukocytes from a healthy volunteer previously immunized with a S. aureus alpha toxoid test vaccine. While the alpha-toxoid used in the Heveker study represents a chemically modified alpha-toxin it could be assumed that the modification does render antigenic determinants less immunogenic or even non-immunogenic and as such the approach could not produce equally effective immunity, as was demonstrated for other bacterial toxins, such as cholera toxin, where toxoid vaccines stimulated anti-toxin antibodies which did not confer immunity to infections (Levine (1983)).
Various factors have been identified in the literature as critical virulence factors for abscess formation, such as toxins, peptidoglycans, extracellular factors and enzymes. A potential role of alpha-toxin in the formation of abscesses was postulated by Kapral et al. (1980). Alpha-toxin is reported to dramatically accumulate in the abscess upon maturation of abscesses although it could not be demonstrated that alpha-toxin is necessary for the abscess formation. A second publication by Adlam et al (1977) negated a key role in the abscess formation for alpha-toxins. The authors demonstrated that alpha-toxin plays a key role in the spreading hemorrhagic form of rabbit mastitis blue-breast seen in natural outbreaks. They reproduced the clinical picture in the laboratory with two unrelated staphylococcal strains. A high circulating anti-alpha-toxin-titer conferred protection against this lethal form of mastitis. Thus, the neutralizing titer could prevent fatal outcome by modifying the clinical picture to the less severe abscess condition. However, neutralization of alpha-toxin did not affect/prevent abscess formation in rabbits. In a more recent publication Kielian et al (2001) investigated the role of alpha-toxin in brain abscess formation in a mouse model. Experimentally the authors implanted wild type S. aureus strains and mutants thereof into the frontal lobe brain tissue and evaluated the ability to induce brain abscesses of each strain. The authors used strains mutant in loci relevant for expression of known virulence factors, e.g. mutants in the sarA locus and the agr locus, both involved in the global regulation of important virulence factors. As alpha-toxin is under control of the sarA/agr regulatory system, the authors also included an alpha-toxin mutant strain into their experiments. The experimental data demonstrated that the replication of mutant bacterial strains for alpha-toxin or the sarA/agr locus had reduced virulence upon injection of bacterial cells into the skull compared to its isogenic control strain RN6390, resulting in lower bacterial numbers and small inflammatory foci in the brains of animals to be detected, as compared to the large well-formed abscesses in those mice receiving the isogenic strain.
However, the mutant strains were not entirely avirulent in the experimental brain abscess model and it cannot be ruled out that additional factor(s) besides alpha-toxin play critical role(s) in brain abscess formation.
The role of alpha-toxin in the abscess formation was evaluated in another experimental setting as outlined by Schwan et al (2003) in an analysis of local, systemic and abscess forming S. aureus infection models. The authors noted that nonhemolytic S. aureus strains became more abundant as time passed in murine abscess and wound models, but not within organ tissues associated with systemic infections. E.g. in a mixed infection using all variants of S. aureus strain RN6390 (hyperhemolytic, hemolytic, and nonhemolytic) in the abscess model, the hyperhemolytic group markedly declined at day 7 post infection, whereas the nonhemolytic population increased significantly. Sequencing of several of the signature-tagged mutants indicated mutations in the agrC gene or within the agrA-agrC intergenic region, which resulted in curtailing both the alpha-toxin and delta-toxin activity. Analyzing specific mutant strains for agr activity (agr-) and alpha-toxin (hla-) in abscess, wound and systemic models of infection, the agr-mutant strain and the hla-mutant strain showed no difference in bacterial counts in murine abscesses at day 4 as compared to the parental wild type strains (RN6390). The same held true for local infections (wound model), whereas considerable clearing of the hla mutant strain and the agr mutant strain occurred in the systemic model of infection. The result clearly indicated the importance of alpha-toxin in systemic infections but not in local infections or abscess formation. In fact mixed infections with the hla-mutant and wild type strains in the abscess model showed a slight advantage given to the hla mutant population over the wild type strain. The authors even concluded that the agr mutations cause reductions in the expression of alpha- and delta-toxins, which in turn contributed to a growth advantage of this agr mutant group within a mixed population of S. aureus cells residing in abscesses and wounds. The results apparently contradict the results described by Kielian et al, where the lack of alpha-toxin production reduced bacterial virulence. Therefore the role of alpha-toxin in abscess formation is not clear.
Overall there is no evidence pointing to one single virulence factor as the main driver in abscess formation. As such research focused on the presence of additional factors not entirely controlled by S. aureus, such as environmental factors, or given structural motifs as the common key factor in abscess formation. E.g. the most recent data regarding virulence factors affecting the formation of abscesses points to the effects of unchelated bivalent metal ions, such as Mn++ and Ca++ on abscess formation and bacterial growth within abscesses. Chelation of metal ions in animals inhibited the formation of liver abscesses and inhibited growth of S. aureus in abscesses (Corbin 2008). On the other hand Tzianabos et al. (2001) hypothesized that an organism such as S. aureus requires virulence factors present on the bacterial cell in order to establish pathological structures such as abscesses in tissue. They demonstrated that strains highly associated with clinical cases of abscesses may possess one or more cell wall-associated polysaccharides with a zwitterionic charge motif (a chemical compound that carries a total net charge of 0, thus electrically neutral but carries formal positive and negative charges on different atoms). In the absence of the zwitterionic charge motif no abscess formation could be observed. The authors concluded that these polysaccharide polymers may modulate abscess induction by this organism. In addition they presented confirming data for not only the core polysaccharides CP5 and CP8 but also for the lipoteichoic acid (LTA), an additional well characterized virulence factor within the cell wall. They identified a zwitterionic charge motif within the LTA as well and therefore generalized their hypothesis for abscess formation to the presence of a zwitterionic charge motif in any pivotal virulence factor for the abscess formation.
Based on the results indicating that various factors contribute to S. aureus mediated abscess formation, a person skilled in the art would not expect that neutralization of a single factor would prevent abscess formation.